Real-time PCR assay for rapid detection of GSTM1 polymorphism in nasopharyngeal carcinoma patients.

Nasopharyngeal carcinoma (NPC) is a common public health problem in Thailand. Glutathione S-transferase M1 gene deletion (GSTM1 null genotype) carriers have been reported to be at increased risk and therefore this parameter is a potential marker for screening of NPC high-risk individuals. However, the conventional polymerase chain reaction (C-PCR) assay commonly used for GSTM1 null genotype detection is not suitable for mass screening since it is inconvenient, time consuming and unsafe due to the use of a toxic chemical. Currently, real-time PCR (R-PCR) assay is recommended for quicker and safer detection of various genetic polymorphisms. The aim of this study was to develop a SYBR green I R-PCR assay combined with melting curve analysis for GSTM1 polymorphism detection in Thai NPC patients. The results were compared to those from the C-PCR assay using DNA samples from peripheral blood leukocytes of 120 Thai NPC patients. The frequencies of GSTM1 polymorphism detected by the R-PCR and the C-PCR were the same. Forty-eight individuals that were GSTM1+ in the R-PCR assay showed 2 peaks with melting points of 82.5 and 87.5 that correlated with the appearance of 2 DNA bands in the C-PCR assay (i.e., one for GSTM1 at 215 base pairs (bp) and one for ?-globin at 268 bp). By contrast, 72 individuals that were GSTM1?- in the R-PCR assay showed 1 peak with a melting point of 87.5C that correlated with the appearance of 1 DNA band for -globin at 268 bp in the C-PCR assay. The R-PCR assay using SYBR Green I and melting curve analysis for GSTM1 polymorphism detection was as reliable as C-PCR assay but was quicker and safer and more amenable to large scale screening in Thai NPC cases.

Detection of GSTM1 polymorphism in patient with nasopharyngeal carcinoma by real-time PCR.

Objective: To investigate whether the real-time polymerase chain reaction (R-PCR) assay with SYBR green I and melting curve analysis could be used for glutathione S-transferase M1 gene (GSTM1) polymorphism detection in Thai nasopharyngeal carcinoma (NPC) patients by comparing the results of this assay with the conventional PCR (C-PCR) assay. Methods: DNA samples from peripheral blood leukocytes of 60 Thai NPC patients were investigated in this study. GSTM1 polymorphism [GSTM1 normal genotype (GSTM1+) and GSTM1 null genotype (GSTM1-)] were examined by using the R-PCR assay with SYBR green I and melting curve analysis and the C-PCR assay. Results: The results of GSTM1 polymorphism detection by the R-PCR assay were in concordance with the C-PCR assay (k = 1.0). Twenty-six individuals with GSTM1+ in the R-PCR assay showed 2 peaks of melting point at 82.5oC and 87.5oC that correlated with the appearance of 2 DNA bands of GSTM1 [215 base pair (bp)] and b-globin (268 bp) in the C-PCR assay, respectively. In addition, thirty-four individuals with GSTM1- in the R-PCR assay showed only 1 peak of melting point at 87.5oC that correlated with the appearance of 1 DNA band of b-globin (268 bp) in the C-PCR assay. Moreover, we found that the R-PCR assay was a faster and safer method for detection of GSTM1 polymorphism than the C-PCR assay. Conclusion: The present study suggests that the R-PCR assay with SYBR Green I and melting curve analysis may be a useful screening tool for more convenient, rapid, reliable, and safer detection of GSTM1 polymorphism in Thai NPC as compared to the C-PCR assay.

Glutathione S-transferase M1 gene polymorphism in Thai nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a serious health problem in Thailand. It is caused by the combined effects of Epstein-Barr virus (EBV), carcinogens and genetic susceptibility. The glutathione S-transferase M1 gene (GSTM1) encodes a phase II enzyme responsible for detoxifying carcinogenic electrophiles. Polymorphic null forms of the gene GSTM1 lack enzyme activity and have been associated with susceptibility to several cancers including NPC. To examine the association between GSTM1 polymorphism and NPC susceptibility in Thais, GSTM1 genotypes (normal and null genotypes) in 78 NPC patients and 145 age-matched healthy controls were determined using PCR assays. Overall, no statistically significant differences were observed in the frequency of GSTM1 genotypes between cases and controls, nor among NPC patients compared on the basis of sex and clinical stage of disease. Carriers with the GSTM1 null genotype had a 2.9-fold increased risk for NPC of WHO type III when compared to those with GSTM1 normal genotype (P < 0.05 with OR =2.9, 95% CI = 1.2-6.8). When cases and controls were categorized into 3 age groups (>40, (>45 and (>50 years), the frequencies of GSTM1 null genotype in cases the (>45 and (>50 age groups were significantly different from controls (P< 0.05). In addition, carriers of the GSTM1 null genotype in age groups (>45 and (>50 years had a 2-fold and 3-fold increased risk for NPC when compared to those with GSTM1 normal genotype (OR = 2.2, 95% CI = 1.1-4.7 and OR = 3.0, 95% CI = 1.2-7.5). We suggest that GSTM1 polymorphism may be associated with NPC susceptibility in Thais, especially for GSTM1 null genotype carriers of age higher than 45 years. The GSTM1 null genotype may be a useful genetic marker for predicting Thai NPC and for screening of early stages of Thai NPC.

Significance of plasma IgA and IgG antibodies to Epstein-Barr virus early and viral capsid antigens in Thai nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is an important causal factor of human nasopharyngeal carcinoma (NPC). High levels of serum IgA and IgG antibodies to EBV early and viral capsid antigens (IgA/EA, IgA/VCA, IgG/EA and IgG/VCA) have been reported in NPC patients. Since specific serum IgA/EA, IgA/VCA and IgG/EA are claimed to be useful serological markers for NPC. In order to evaluate whether plasma IgA/EA, IgA/VCA, IgG/EA and IgG/VCA antibody levels are useful markers for diagnosis and prognosis of Thai NPC, we examined the prevalence of these antibodies in 79 NPC patients, and 127 age-matched controls (47 healthy subjects (HS), 32 cases of other disease (OD) and 48 cases of other cancer (OC)) by using an indirect immunofluorescence assay. The prevalence of plasma IgA/EA, IgA/VCA, and IgG/EA in NPC patients (55.7, 68.4 and 68.4%) was significantly higher than in the HS (0.0, 0.0 and 20.5%,), OD (0.0, 0.0 and 3.1%) and OC (0.0, 0.0 and 20.8%) groups (p<0.05). The prevalence of plasma IgG/VCA in NPC patients (93.7%) was significantly different from those for the OD and OC groups (71.9 and 43.8%) but not for the HS group (89.4%). In NPC patients, the geometric mean titers (GMT) of plasma IgA/EA, IgA/VCA and IgG/EA were increased with an advanced clinical stage of disease but not IgG/VCA. In contrast, GMT of IgG/VCA was increased with aggressive type of disease (histological type) but not IgA/EA, IgA/VCA, and IgG/VCA. The results of our study suggest that plasma IgA/EA, IgA/VCA and IgG/EA antibodies may be useful markers for diagnosis and assessing prognosis of Thai NPC.